The host plays a role not only in replication of plus-stranded RNA viruses, but also in viral RNA recombination, although the role(s) of host factors in recombination is currently unknown. RNA recombination is an important mechanism for plus-strand RNA viruses, because it contributes to the emergence of new pathogenic or drug-resistant viruses and strains. Accordingly, RNA recombination has been demonstrated to occur for a large number of human, animal and plant viruses, including poliovirus, dengue virus, infectious bronchitis virus, influenza A virus, hantavirus, hepatitis D virus, Human Respiratory Syncytial Virus, Corona-, Rota-, Flavi-, Calici-, Arteri-, Astro-, Entero-; and Alphaviruses. In order to advance understanding of the role of the host in viral RNA recombination, the PI has recently developed a yeast-based recombination system for a model plant virus, Cucumber necrosis tombusvirus (CNV). The CNV replicase is similar to the replicases of important human and animal viral pathogens, such as hepatitis C virus, Flaviviruses and Pestiviruses. CNV is exceptionally suitable for studying RNA recombination because numerous recombinants can easily and rapidly be recovered: (i) in yeast cells, (ii) in whole plants, and (iii) in the in vitro CNV RNA-dependent RNA polymerase (RdRp) assay. The central hypothesis tested in this work is that host factors may affect viral RNA recombination. The PI will use (Aim 1 and 2) the advanced genetics tools available for yeast, a model host. Encouraged by preliminary data on the efficient replication and recombination of a Tombusvirus replicon in yeast, the PI will use a high-throughput screen to test the effect of each host gene on viral RNA recombination based on the yeast deletion strain library (~ 5,500 strains), in which each yeast gene was separately deleted. A partial screen of the library has already identified three host genes affecting RNA recombination, thus confirming the feasibility of the approach. The identified host genes affecting viral RNA recombination will be further tested (Aim 3) to characterize their effect on viral recombinants. The research described here will lead to better understanding of the role of host genes in viral RNA recombination and the role of the host in virus evolution. Knowledge of recombination promises untold health benefits by providing means to limit virus infections and to develop safer vaccines.